The human heat shock protein 70 (Hsp70) family is evolutionarily conserved among all organisms from archaebacteria to humans, suggesting an essential role in cell survival (1, 2). Under circumstances of transient cell stress, the heat shock response and activities of molecular chaperones can restore protein homeostasis. In human disease, however, misfolded proteins can accumulate when polyglutamine-expansion proteins are chronically expressed over the life of the cell. Elevated expression of molecular chaperones suppresses protein misfolding/aggregation and toxicity phenotypes in various model systems of Huntington's disease, Alzheimer's disease, Parkinson's disease, and Amyotrophic Lateral Sclerosis (ALS). Mutations in the respective proteins huntingtin, tau, alpha-synuclein, and superoxide dismutase (SOD1), associated with these diseases, result in the appearance of misfolded species that adopt alternate conformations. These observations led to the proposal that a common feature of diverse diseases of protein conformation is the appearance of alternate folded states that self-associate and form toxic species and protein aggregates.

A role for Hsp70 family proteins in controlling these events has been widely studied. Studies with mammalian tissue culture cells, transgenic mice, Drosophila, and C. elegans have established that the heat shock response can be activated in cells expressing aggregation-prone proteins, suggesting a role for molecular chaperones as an adaptive survival response (3, 4). Moreover, a direct relationship with polyglutamine diseases is suggested by the co-localization of several heat shock proteins, including Hdj-1, Hdj-2, Hsp70 and ubiquitin with polyglutamine aggregates in the tissues of affected individuals, transgenic mice and tissue culture cells (5). Finally, overexpression of Hsp70 can suppress the toxicity associated with the accumulation of misfolded proteins (6-8). High throughput screening initiatives aimed at the identification of compounds that enhance the heat shock response, in particular Hsp70, will provide insights into this conserved cellular process and may lead to novel therapeutics for these devastating disorders.

Assay Overview:The purpose of this assay is to determine whether powder samples of a compound identified as transcriptional activators of heat shock protein 70 (Hsp70) modulates the gene expression of Hsp70 involved in the heat shock response and protein homeostasis. In this assay, HeLa cells are incubated with test compound and harvested. RNA is purified and subjected to quantitative reverse transcription (qRT-PCR) of Hsp70 and GAPDH (control). Gene expression is normalized to GAPDH and compared to levels in cells treated with DMSO only. As designed, test compounds that induce an increase in Hsp70 gene expression will result in an increase in amplified RNA product.Protocol Summary:HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with phenol red buffered with HEPES and supplemented with 10% v/v fetal bovine serum (FBS), 1% L-glutamine, and 100 U/ml penicillin/streptomycin. The cells were treated with test compound, the positive controls celastrol (3 uM), CdCl2 (50 uM) and MG132 (10 uM), or left in vehicle (DMSO). The cells were harvested 4 hours after compound addition for analysis of chaperone expression by quantitative reverse transcription (qRT)-PCR. RNA was purified using the RNeasy Mini kit according to the manufacturer's instructions. After the reverse transcription reaction, PCR was performed using PCR primers specific for human Hsp70 and GAPDH. PCR products were amplified with Taq polymerase by using standard cycling conditions. Gene expression was normalized to GAPDH and compared to levels in cells treated with DMSO (mRNA levels set as 1.0) using ImageJ.PubChem Activity Outcome and Score:Compounds that induced a minimum of 1.5-fold induction in gene expression compared to the DMSO levels (set at 1.0) were active in this assay.The PubChem Activity Score is assigned a value of 100 for active compounds, and 0 for inactive compounds.The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.List of reagents:HeLa cells (provided by Assay Provider)DMEM (Invitrogen)RNeasy Mini kit (Qiagen, part 74106)Taq polymerase (Promega, part M3001)PCR primers were ordered as appropriate. The primer pair sequences for the human Hsp70 and GAPDH gene targets are indicated below:Hsp70 forward: 5'-AGAGCCGAGCCGACAGAG-3'Hsp70 reverse: 5'-CACCTTGCCGTGTTGGAA-3'GAPDH forward: 5'-GTCGGAGTCAACGGATT-3'GAPDH reverse: 5'-AAGCTTCCCGTTCTCAG-3'